1. Field of the Invention
The present invention relates to a method for producing a receptor chip by immobilizing a biotinylated recombinant receptor protein on a chip, a detection kit comprising the receptor chip, and a receptor chip produced by the method.
The present invention relates to a receptor chip produced by expressing a region of a scavenger receptor relating to a ligand recognition site which is used as a receptor fragment, in vivo or in vitro, and immobilizing the fragment on a solid phase. The receptor chip is useful as a high-sensitivity receptor chip for detection of modified LDL (Low Density Lipoprotein), such as oxidized LDL, acetylated LDL, succinylated LDL, and malondialdehyde LDL, glycosylated LDL, or the like, abnormal cells, and bacteria.
The present invention relates to a method and kit for detecting modified LDL, abnormal cells (e.g., apoptotic cells, etc.), or bacteria. To obtain this invention, an extracellular region and a C-type lectin-like domain (CTLD) of a scavenger receptor is expressed in cells (e.g., E. coli or the like) or in test tubes as biotinylated proteins. Thereafter, the erroneous structure of an inactive aggregate of biotinylated protein is unfolded with a denaturant. The protein is refolded with a detergent and a cyclic carbohydrate into a correct higher-order structure. The resultant protein is immobilized via avidin, streptavidin, or the like, on solid phase while keeping an appropriate direction thereof. The thus-obtained solid phase is employed as a sensor portion (detection sensor) in an intermolecular interaction analysis method, for example, a detection method using a detector, such as, a surface plasmon resonance device, a quartz-crystal microbalance device, or the like.
According to the present invention, it is possible to produce a receptor chip on which any receptor or a receptor fragment is immobilized.
2. Description of the Related Art
Receptors present on a cell surface specifically bind with ligands corresponding to the receptors. As a result, various signals are transduced into cells. There are a variety of receptors present on a cell surface and the corresponding receptors are different from one another. Therefore, in order to detect and/or quantitate a specific ligand, it is useful to use a receptor capable of being specifically binding with the ligand. If a receptor chip is produced whose ligand is a diagnostic marker for abnormal cells or diseases, it is expected to provide a tool useful for the detection of abnormal cells in a cell population or for the diagnosis of diseases.
For example, a plurality of receptors capable of recognizing and binding modified LDL accumulated in an organism, and abnormal cells such as apoptotic cells or aged erythrocytes, bacteria invading an organism, or the like have been found. Among these receptors, there are a number of receptors whose region required for recognition of a ligand has been conjectured. There is a possibility that by employing these receptors themselves or only regions thereof required for recognition, ligands, i.e., modified LDL, abnormal cells (e.g., apoptotic cells, etc.), and bacteria can be easily detected.
There is a demand for the production of a high-sensitivity ligand sensor in which a region of a receptor relating to the ligand recognition site is immobilized on a solid phase, such as a chip, for ligand recognition utilizing a receptor.
However, receptor proteins are generally expressed in a small amount. Therefore, it is difficult to prepare a large amount of a native receptor protein to such an extent that a receptor chip can be produced. Further, when a recombinant expression system is employed, since a receptor is a membrane protein, a region essentially required for recognition needs to be obtained as a soluble protein in order to construct a system capable of detection. In addition, the thus-obtained soluble protein needs to be modified so that it can serve as a sensor capable of detection.
However, in conventional techniques, it is difficult to obtain a soluble protein itself. A most simple, efficient, and inexpensive method for obtaining a soluble protein is to express a protein of interest using genetic engineering techniques, particularly using E. coli as a host. However, with this method, expression products are accumulated in bacteria as an inactive aggregate called an inclusion body, and therefore, it is not possible to obtain a soluble protein.
Animal cells may be employed as hosts to obtain a soluble protein useful for measurement of a ligand. For example, Japanese Laid-Open Publication No. 2002-17353 discloses a method for quantitating modified LDL using a receptor. Specifically, an animal cell is used to express a fusion protein comprising an extracellular region of an oxidized LDL receptor and a part of the constant region of an immunoglobulin heavy chain. By detecting the fusion protein with an immunoassay, an oxidized LDL can be quantitated with high sensitivity. However, such a method using animal culture cells requires much labor and cost.
An attempt has been made to refold an aggregate derived from a receptor protein accumulated in E. coli into a soluble correct structure (e.g., Japanese Laid-Open Publication No. 2003-169693 relating to soluble IL-15 receptor α chain). However, in conventional methods, for example, after a receptor is adsorbed onto a resin, the resultant resin is contacted with a buffer solution containing a denaturant and then with buffer solutions having a gradually reduced concentration of a denaturant (Japanese Laid-Open Publication No. 2003-169693). Thus, conventional methods are complicated. Further, when a protein is refolded as it is immobilized on a solid phase, the step of eluting or cleaving the protein from the solid phase, or the like, is required after refolding, resulting in an increase in method complexty, a reduction in yield, and the like. Furthermore, the obtained soluble protein was not appropriately modified for use in a detection system no matter whether a solid phase was used. It was not possible to immobilize a refolded protein at a desired position.
It is known that addition of a His tag or a GST tag, or biotinylation of an expression product are used for immobilization of proteins based on affinity. Among these techniques, protein biotinylation is preferable because of less steric hindrance and less influence from metal ions and reducing agents. Biotinylation needs to be carried out within bacteria in order to achieve efficient biotinylation without impairing the function of expression products. However, conventional techniques have difficulty in refolding products expressed under conditions which permit generation of inclusion bodies, and a low expression level and degradation of products expressed under conditions that do not permit generation of inclusion bodies. Therefore, a receptor chip has not been produced by expression and immobilization of biotinylated receptor proteins.
For the above-described reasons, a method has not been currently established to produce a receptor, which can be easily immobilized on a solid phase while keeping the ability of being bound to a ligand.